The present invention relates to a vector which can express the full-length gene of RNA virus, an animal cell and RNA virus infected animal model which comprise the vector, and a method for screening an agent using the cell and the animal model. These are useful to elucidate the mechanisms of RNA virus replication and the development of RNA virus infections, and to develop therapeutic agents and means of treatment, and so on.
Hepatitis C virus (hereinafter, HCV) is a principal causative virus in the, development of non-A, non-B post-transfusion hepatitis (Saito, I. et al., Proc. Natl. Acad. Sci. USA, 87, 6547-6549 (1990)). Since the hepatitis caused by this virus has high chronicity and may often develop into hepatocirrhosis or hepatoma, this is one of the infections for which the invention of a reliable means of treatment is urgently required. The cDNA of this virus was cloned by Choo et al. in 1989 (Choo, Q. -L., et al., Science, 244, 359-362 (1989)), and it is known that the virus is a single-stranded RNA virus belonging to the family Flaviviridae (Kato, N., et al., Proc. Natl. Acad. Sci., USA, 87, 9524-9528 (1990)). Several study groups have clarified its total nucleotide sequence and amino acid sequence (Kato, N., et al., Proc. Natl. Acad. Sci., USA, 87, 9524-9528 (1990), Proc. Natl. Acad. Sci., USA, 88, 2451-2455 (1991), J. Virol., 65, 1105-1113 (1991), J. Gen. Virol., 72, 2697-2704 (1991), Virology, 188, 331-341 (1992)).
Although there are several reports on the establishment of an in vitro infection system of HCV, the real picture is that a stable infection system with repeatability which is practical enough to be utilized for the elucidation of various mechanisms of the virus or the development of means of treatment has not yet established due to problems including low reproduction amounts. (Lanford, R E, et al., Virology, 202, 606(1994), Yoo, B J, et al., J. Virol., 69,32(1995), Shimizu, Y K, et al., Proc. Natl. Acad. Sci. USA, 89, 5477(1992), Kato, N., et al., Biochem. Biophys. Res. Comm., 206, 863(1995), Batolini, L., et al., Res. Virol., 144,281 (1993)).
On the other hand, there is another method for generating HCV, wherein first an RNA viral genome is generated by transcribing from the corresponding cDNA and then the virus is generated via protein synthesis (Raccaniello, V R, Science, 214, 916(1981), Poliomyelitis virus). Regarding this method also, several study groups have been working energetically, but there has not been found any practical system, just as in the case of the above-stated infection system (Mizuno, M, et al., Gastroenterology, 109, 1933(1995), Dash, S., et al., Am. J. Pathol., 151,363 (1997)). Furthermore, regarding small animals expressing full-length cDNA such as a transgenic mouse, it has been reported that there is a mouse expressing a part or whole cDNA, but animals which can effectively express all viral proteins are still unknown (Japanese Patent Application Laying-Open (kokai) Nos. 9-9965 and 10-84813).
From the study of the present inventors, the reason why an effective amount of viral particles or viral proteins cannot be generated is considered to be that both termini of the viral genome generated in the process are not transcribed properly so that replicable full-length genes are not generated effectively.
The objects of the present invention are to construct an expression system generating a full-length virus genome, to build up an expression system which resembles more closely the replication of the original virus, and to establish cells or animal models expressing a virus from cDNA using the system.
After thorough study directed to achieve the above objects, the present inventor has succeeded in constructing a vector which can express a full-length RNA viral gene and establishing a cell strain into which the vector is integrated, thereby finally completing the present invention.
That is to say, the first aspect of the present invention relates to a vector which comprises cDNA encoding an RNA viral gene, and the vector is characterized in that it is constructed such that both termini of the RNA viral gene can be transcribed precisely and uniformly.
The second aspect of the present invention relates to an animal cell which is characterized in that it comprises the vector.
The third aspect of the present invention relates to an RNA virus infected animal model which is characterized in that it comprises the vector in a cell thereof.
The forth aspect of the present invention relates to a method for screening an agent inhibiting the replication of an RNA virus wherein the animal cell or the RNA virus infected animal model is used.
This specification includes the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 10-177820 which are priority documents of the present application.
The details of the present invention are disclosed below.
(1) The First Invention (a Vector)
The vector of the present invention comprises cDNA encoding an RNA viral gene, and is characterized in that it is constructed so that both termini of the RNA viral gene can be transcribed precisely and uniformly. The description xe2x80x9cboth termini . . . can be transcribed preciselyxe2x80x9d in this specification means that an RNA produced from cDNA is completely identical to a genomic RNA derived from the virus itself or does not present any differences in nucleotide sequence great enough to influence translation ability. On the other hand, xe2x80x9cboth termini . . . can be transcribed uniformlyxe2x80x9d means that the vector can produce with a certain repeatability an RNA having specific nucleotide sequences.
RNA viruses which can be used for the present invention include, but are not limited to, picornavirus such as poliovirus, coxsackievirus and echovirus; reovirus; togavirus including flavivirus such as HCV; orthomyxovirus; paramyxovirus; coronavirus; or plant RNA virus such as tobacco mosaic virus. A preferable RNA virus is HCV.
Methods of transcribing both termini of an RNA viral gene include, but are not limited to, a method wherein DNA encoding a ribozyme which is capable of cleaving the RNA viral gene by self-processing is positioned both upstream of the 5xe2x80x2-terminus and downstream of the 3xe2x80x2-terminus of the cDNA encoding the RNA viral gene.
Ribozymes cleaving an RNA viral gene by self-processing include xcex4 type hepatitis virus (Hepatitis Delta Virus: HDV) ribozyme, hammerhead ribozyme, hairpin ribozyme, artificial ribozyme obtained from in vitro and in vivo selections and the like. Regarding the nucleotide sequence of each of the above ribozymes, there is the description by Eiko Ootsuka et al., Protein, Nucleic Acid and Enzyme 40, 1400 (1995). In particular, the nucleotide sequence of HDV ribozyme is disclosed by Suh, Y-A., et al., Nucleic Acids Research, 20, 747(1992), and that of hammerhead ribozyme is disclosed by Shimayama, T., et al., Biochemistry, 34, 3649(1995). The DNA encoding the ribozyme positioned in a vector can be determined according to the kind and nucleotide sequence of the RNA virus to be used, with reference to the common nucleotide sequences described in these reference documents. Taking HCV as an example, a DNA encoding hammerhead ribozyme at the 5xe2x80x2-terminus side (see sequence X set forth below as an example) and a DNA encoding HDV ribozyme at the 3xe2x80x2-terminus side (see sequence Y set forth below as an example) are considered to be preferable.
SEQ ID NO: X
CTGATGAGGCCGAAAGGCCGAAACGGCGAAAGCCGTC (SEQ ID NO: 7)
SEQ ID NO: Y
TGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATTCCGA OGGGACCGTCCCCTCGGTAATGGCGAATGGGAC (SEQ ID NO: 8)
The vector of the present invention can be produced by, firstly preparing a DNA fragment containing two DNAs encoding the above-described ribozyme and a DNA encoding an RNA virus by PCR or the like, and secondly inserting the DNA fragment into a vector containing a proper promoter and terminator.
The vector of the present invention may express immediately after it was transferred into a host cell, but it is more preferable that the vector initiates expression by a specific treatment. The methods for effecting the initiation of expression by a specific treatment include a method of using a promoter which is not recognized by RNA polymerase of the host cell, a method using Cre/lox expression system (Nat sternberg et al. J. Molecular Biology 150. P467-486, Japanese Patent Application Laying-Open (kokai) No. 10-84813) and so on. In the former, the expression of the gene of interest can be initiated by expressing RNA polymerase, which can recognize a promoter in a vector, within the host cell. In the latter method, the expression of the gene of interest can be initiated by expressing Cre enzyme in the host cell.
(2) The Second Invention (an Animal Cell)
The animal cell of the present invention is characterized in that it comprises the vector of the present invention (the first invention).
The animal cell of the present invention can be produced by transferring the vector of the present invention into an animal cell used as a host. Host animal cells include, but are not limited thereto, IMY, HuH-7, HepG2, MOLT-4, MT-2, Daudi, a hepatic primary cell, other hepatocytes, a cell and cell strain derived from hemocyte system cells and the like. The methods for transferring the vector into the host include Lipofection Reagent, but are not limited to this.
(3) The third Invention (an RNA Virus Infected Animal Model)
The RNA virus infected animal model of the present invention is characterized in that it comprises the vector of the present invention in a cell thereof.
The RNA virus infected animal model of the present invention is prepared by a process of transferring the vector of the present invention into zygotes which are then transferred to false parents, obtaining animals derived from the zygotes, and selecting therefrom individuals expressing an RNA virus gene which has integrated.
The introduction into a zygote can be carried out in accordance with standard techniques such as microinjection. The target animals include any of those on which the production technique of the transgenic animal has been established, including mouse, rat, rabbit, pig, cyprinodontidae, zebra fish etc. The individual expressing an RNA virus gene is selected, e.g. by PCR or the like wherein primers prepared on the basis of nucleotide sequences specifically existing in the RNA virus gene are used.
(4) The Forth Invention (a Method for Screening an Agent)
The method for screening an agent inhibiting the replication of an RNA virus of the present invention is characterized in that the animal cell of the present invention (the second invention) or the RNA virus infected animal model of the present invention (the third invention) is used.
When the animal cell is used, the screening can be carried out by adding the target agent to a medium. When the animal model is used, it can be carried out by administering the target agent intravenously or orally.